Alternative splicing regulates vesicular trafficking genes in ...

NIH Public Access Author Manuscript Nat Commun. Author manuscript; available in PMC 2014 October 22.

NIH-PA Author Manuscript

Published in final edited form as: Nat Commun. ; 5: 3603. doi:10.1038/ncomms4603.

Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development Jimena Giudice1, Zheng Xia2,3, Eric T. Wang4,5, Marissa A. Scavuzzo1, Amanda J. Ward1,2, Auinash Kalsotra1, Wei Wang6, Xander H.T. Wehrens6,7, Christopher B. Burge4, Wei Li2,3, and Thomas A. Cooper1,2,5,* 1Department

of Pathology and Immunology, Baylor College of Medicine (BCM), Houston, TX

77030, USA 2Department

of Molecular and Cellular Biology, Baylor College of Medicine (BCM), Houston, TX

77030, USA


3Division

of Biostatistics, Dan L. Duncan Cancer Center, Baylor College of Medicine (BCM), Houston, TX 77030, USA 4Department

of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

5Koch

Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

6Department

of Molecular Physiology and Biophysics, BCM, Houston, TX 77030, USA

7Department

of Medicine, BCM, Houston, TX 77030, USA

Abstract


During postnatal development the heart undergoes a rapid and dramatic transition to adult function through transcriptional and post-transcriptional mechanisms, including alternative splicing (AS). Here we perform deep RNA-sequencing on RNA from cardiomyocytes and cardiac fibroblasts to conduct a high-resolution analysis of transcriptome changes during postnatal mouse heart development. We reveal extensive changes in gene expression and AS that occur primarily between postnatal days 1 and 28. Cardiomyocytes and cardiac fibroblasts show reciprocal regulation of gene expression reflecting differences in proliferative capacity, cell adhesion

*

Correspondence: Thomas A. Cooper, MD. Department of Pathology and Immunology, Baylor College of Medicine, One Baylor Plaza, room 268B. Houston, TX 77030, USA. [email protected]. Phone: 1-713-798-3141. Fax: 1-713-798-5838. Present addresses: Isis Pharmaceuticals, Carlsbad, CA 92010, USA (AJW). Departments of Biochemistry and Medical Biochemistry, University of Illinois, Urbana-Champaign, IL 61801, USA (AK). AUTHORS´ CONTRIBUTIONS JG designed research, performed the experiments, analyzed the data and wrote the manuscript. ZX, WL, performed computational analysis of sequencing reads from RNA-seq data, iCLIP and motif analysis of splicing events, and contributed to the manuscript. ETW, CBB performed computational analysis of sequencing reads from RNA-seq data and contributed to the manuscript. MAS performed RT-PCR validations and contributed with manuscript suggestions. AJW isolated CELF1-expressing hearts and controls for RNA-seq. AK provided suggestions for initial cell isolation set-up and contributed to the manuscript. WL, XHTW collaborated in Ttubule/calcium experiments. TAC supervised and designed research, analyzed the data and wrote the manuscript.

COMPETING FINANCIAL INTERESTS NONE ACCESSION Codes RNA-Seq data have been deposited at NCBI Gene Expression Omnibus under accession code GSE49906.

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functions, and mitochondrial metabolism. We further demonstrate that AS plays a role in vesicular trafficking and membrane organization, These AS transitions are enriched among targets of two RNA-binding proteins, Celf1 and Mbnl1, which undergo developmentally regulated changes in expression. Vesicular trafficking genes affected by AS during normal development (when Celf1 is down-regulated) show a reversion to neonatal splicing patterns after Celf1 re-expression in adults. Short-term Celf1 induction in adult animals results in disrupted transverse tubule organization and calcium handling. These results identify potential roles for AS in multiple aspects of postnatal heart maturation, including vesicular trafficking and intracellular membrane dynamics. The heart is the first organ to form and function during vertebrate embryogenesis1. The first four postnatal weeks involve a period of extensive physiological remodeling with dynamic changes as the fetal heart adapts to birth and converts to adult function. This transition occurs through transcriptional and post-transcriptional mechanisms, including coordinated networks of alternative splicing (AS)1–4.


Human and rat hearts are composed of 66% cardiac fibroblasts (CF), 30% cardiomyocytes (CM), and 4% endothelial and vascular smooth muscle cells5–7. Studies differ regarding adult mouse heart composition. While Soonpaa et al. reported that CF account for 86% of cells8, a recent analysis demonstrated a composition of 26% CF, 56% CM, and 18% nonCM and non-CF9. However, CM comprise ~75% of the tissue volume in mammals7. CM generate the contraction force and CF form the mechanical scaffold required for effective pumping10. CM and CF communicate through multiple signaling mechanisms and through extracellular-matrix (ECM)11. Other CF functions include response to cardiac injury12 and electrical isolation of different regions of the cardiac conduction system13. By postnatal day 7 (PN7), CM lose proliferative capacity and heart size increases due to CM hypertrophy14–15. Limited microarray analysis of mRNA expression in freshly isolated CM and CF showed that while certain genes are highly expressed in CM, many growth factors, cytokines, and ECM genes are more highly expressed in CF16. Overall, the published data address a limited number of gene expression changes in CM and CF during development, and notably, do not provide AS information.


High-throughput studies of AS and gene expression regulation have primarily focused on differences between tissues, normal versus pathological conditions, or cultured cells. A small set of reports have addressed AS and gene expression changes during normal physiological transitions17–21. Development provides an outstanding opportunity to identify coordinated AS regulation critical for physiological transitions from embryonic to adult functions. Previously, we showed that genes that undergo AS regulation during heart development produce transitions from embryonic to adult protein isoforms largely without changes in overall transcript levels, presenting a new paradigm for understanding developmentally regulated gene expression in heart3. Nearly half of the AS transitions identified in mouse are conserved during post-hatch chicken heart development, suggesting highly conserved functions for splicing-mediated isoform transitions3. In the present study, we analyzed AS and gene expression transitions regulated during postnatal mouse heart development using mRNA deep sequencing (RNA-seq)22. To gain insight into the diversity of cell type-specific transitions, we performed RNA-seq using Nat Commun. Author manuscript; available in PMC 2014 October 22.

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freshly isolated CF and CM from a developmental time course. The results revealed that most gene expression and AS changes occurs within the first four weeks after birth, and that CM and CF exhibit reciprocal transitions in expression of specific functional categories (proliferation, cell adhesion, cytokines-chemotaxis, metabolism, transcription regulation). Interestingly, we found that genes involved in vesicular trafficking and membrane organization are regulated by AS during postnatal CM development. These AS changes are enriched as targets of the CUGBP, ELAV-Like family (Celf) and Muscleblind-like (Mbnl) RNA-binding protein families, both of which are involved in AS and are regulated during postnatal heart development3,23–24. In the heart, vesicular trafficking-related AS transitions likely impact ligand/growth factor uptake, ion channels dynamics, and/or postnatal formation of the sarcoplasmic reticulum (SR) and transverse tubules (T-tubules), crucial processes for excitation-contraction coupling (ECC) that are established by PN3025. We show that re-expression of CELF1 in adults specifically in CM results in altered T-tubule structure and mis-regulated calcium handling consistent with alterations associated with reexpression of fetal splicing patterns.

RESULTS NIH-PA Author Manuscript

Extensive transcriptome changes during postnatal development RNA-seq was performed using RNA from mouse ventricles isolated at five time points: E17, PN1, PN10, PN28, and adult (PN90). cDNA libraries were prepared after ribosomal RNA (rRNA) depletion for 100 bp paired-end reads using the Illumina HiSeq2000. We obtained >150 million read pairs per sample, >80% of which mapped the mouse genome (Supplementary Table 1).


We identified 2,568 differentially expressed genes (≥2.0 fold, FDR 0.01, Supplementary Material) between E17-adult: 747 were up-regulated and 1,821 down-regulated (Fig. 1a). Analysis of mRNA isoforms whose percent spliced in (PSI)26 values changed ≥20% (ΔPSI≥20%) between E17-adult identified 927 AS events, 190 alternative 3´ untranslated regions (UTRs), and 210 alternative first exons (Fig. 1b). Postnatal AS transitions were predominantly cassette exons (62%), while alternative 3´ and 5´ splice sites each represented 9%. We observed a relatively high proportion of transitions (20%) involving intron retention with roughly equal proportions of events exhibiting increased inclusion or exclusion of the variable regions during development (Fig. 1c–d). Extensive remodeling within the first four weeks after birth Gene ontology (GO) analysis of down-regulated genes between E17-adult in ventricles showed a clear enrichment (p